A microtranslatome coordinately regulates sodium and potassium currents in the human heart

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  Figure 1.. Complex of ion channel transcripts with nascent proteins. (A) Scheme of the RNA-IP protocol in which channel-specific antibodies are used to pull down nascent proteins and associated transcripts. RNP: ribonucleoprotein. (B) Lanes 1 and 2, RT-PCR products from input lysate of human left ventricle (LV), and iPSC-CM. Lanes 3–16 shows the corresponding RNA-IP’s using an anti-hERG1a or anti-NaV1.5 antibodies; Lane seven shows the control (+) and represents signal amplified from purified plasmid template. Similar results were obtained in at least three independent experiments. (N = 5 for anti-hERG1a and N = 3 for anti-Nav1.5 using human LV and iPSC-CMs). 10.7554/eLife.52654.004Figure 1—source data 1.RNA-IP Blots raw data for Figure 1B.

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  Figure 1—figure supplement 1.. Complete RNA-IP from Figure 1. Lanes 1–6, RT-PCR products from input lysate of human left ventricle (LV), iPSC-CM, and HEK293 cells expressing: hERG1a; SCN5A; hERG1a plus SCN5A; and hERG1a plus hERG1b and SCN5a. Lane seven shows RT-PCR product from lysates independently expressing hERG1a and SCN5A, mixed. Lanes 8–14 shows the corresponding RNA-IP’s using an anti- hERG1a antibody, followed by a bead-only control and H2O control. The next group shows the corresponding RNA-IP’s using the anti-Nav1.5 antibody, followed by a group of IgG controls. H2O and beads lanes show absence of template contamination; control (+) represents signal amplified from purified plasmid template.

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  Figure 2.. Quantitative description of single hERG1a and SCN5A transcripts and their distribution in iPSC-CMs. (A) Representative confocal images and enlargement (outlined in yellow) of iPSC-CMs subjected to the smFISH protocol. (B) By fitting the intensity histogram of smFISH signals (n = 2611 spots) to the sum of Gaussian functions (red line), the typical intensity corresponding to a single mRNA molecule (vertical dashed line) was extracted. (C) The distribution of the number of mRNA molecules associated in clusters for each transcript evaluated by smFISH. (D) Histogram showing the cytoplasmic distribution of mRNA signals with distance from the nucleus. (E) The number of mRNAs detected per cell was plotted for SCN5A, hERG1a and GAPDH (lines represent mean ±SE). 10.7554/eLife.52654.010Figure 2—source data 1.Intensities plot for determination of single mRNA intensity raw data for Figure 2B. 10.7554/eLife.52654.011Figure 2—source data 2.Clusterization of transcripts raw data for Figure 2C. 10.7554/eLife.52654.012Figure 2—source data 3.Distance of mRNA from the nucleus raw data for Figure 2D. 10.7554/eLife.52654.013Figure 2—source data 4.Numbers of mRNA per cells raw data for Figure 2E.

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  Figure 2—figure supplement 1.. Specificity of the probes used in smFISH experiments. Representative images of smFISH for either hERG1a (top panel) or SCN5A (bottom panel) mRNAs performed in HEK293 cells transiently transfected with hERG1a or SCN5A. Only the cells expressing hERG1a or SCN5A showed a positive signal for smFISH revealing the specificity of the probes used in smFISH experiments.

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  Figure 2—figure supplement 2.. Single mRNA intensity determination. The distribution of total fluorescence intensity of smFISH signals for hERG1a (2611 spots; A), SCN5A (2815 spots; B), and GAPDH (3507 spots; c). By fitting the histogram to the sum of Gaussian functions (red line), the typical intensity corresponding to a single mRNA molecule (vertical dashed line) was extracted. 10.7554/eLife.52654.008Figure 2—figure supplement 2—source data 1.Single mRNA intensity determination for Figure 2—figure supplement 2.

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  Figure 2—figure supplement 3.. Quantification of mRNA expression using two different methods. (A) The distribution of total fluorescence intensity of smFISH signals for hERG1a (2892 spots) obtained using FISHQUANT software for analysis. By fitting the histogram to the sum of Gaussian functions (red line), the typical intensity corresponding to a single mRNA molecule (vertical dashed line) was extracted. (B) Comparison of the number of mRNA molecules detected per cells for hERG1a using two different methods of analysis (Method 1: manual using ImageJ; Method 2: Semi-automatic using FISHQUANT).

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  Figure 3.. hERG1a and SCN5A transcript expression levels correlate. (A) Representative confocal images and enlargements of double smFISH experiments for SCN5A (red) and hERG1a (cyan) mRNAs. (B) The number of mRNA molecules detected per cell in double smFISH experiments were plotted for SCN5A and hERG1a and the coefficient of determination R2 was determined from the Pearson’s correlation coefficient R (n = 41 cells; N = 2). (C) Representative confocal images and enlargements of double smFISH experiments for RyR2 (red) and hERG1a (cyan) mRNAs. (D) The number of hERG1a mRNA was plotted against the number of RYR2 mRNAs per cells and showed a low correlation in their expression (n = 26 cells; N = 2). 10.7554/eLife.52654.017Figure 3—source data 1.Correlation analysis of hERG1a and SCN5A mRNA expressions raw data for Figure 3B. 10.7554/eLife.52654.018Figure 3—source data 2.Correlation analysis of hERG1a and RyR2 mRNA expressions raw data for Figure 3D.

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  Figure 3—figure supplement 1.. Correlation of mRNA expression. The number of mRNA molecules detected per cells in double smFISH experiments were plotted for SCN5A and GAPDH (28 cells, (A), and hERG1a and GAPDH (13 cells, (B). The Pearson’s correlation coefficient (R2) were calculated for each pairs of mRNAs. 10.7554/eLife.52654.016Figure 3—figure supplement 1—source data 1.Correlation of mRNA expression for Figure 3—figure supplement 1.

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  Figure 4.. hERG1a and SCN5a transcript colocalization. (A) Representative confocal images and enlargement (outlined in yellow) of iPSC-CMs subjected to smFISH showing the colocalization of hERG1a and SCN5A mRNAs. (B) Comparison of the average number of associated hERG1a and SCN5A mRNAs particles observed vs. expected by chance using different overlap criteria illustrated (mean ±SE; n = 41 cells; N = 2). (C) Diagram illustrating that the association of hERG1a and SCN5A mRNAs account for 24% and 23% of their total population respectively. (D) Representative confocal images of smFISH for hERG1a and RyR2 transcripts. (E) Comparison of the average number of associated hERG1a and RyR2 mRNAs particles observed vs. expected by chance using different overlap criteria (mean ±SE; n = 26 cells; N = 2). 10.7554/eLife.52654.020Figure 4—source data 1.Association of hERG1a and SCN5A transcripts raw data for Figure 4B. 10.7554/eLife.52654.021Figure 4—source data 2.Proportion of hERG1a and SCN5A mRNA association raw data for Figure 4C. 10.7554/eLife.52654.022Figure 4—source data 3.Association of hERG1a and RyR2 transcripts raw data for Figure 4E.

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  Figure 5.. Cotranslational association of hERG1a protein and hERG1a and SCN5A mRNAs. (A) Representative confocal images and enlargement of iPSC-CMs subjected to immunofluorescence (IF) combined with smFISH protocol. Arrows indicate triply colocalized particles. (B) The average number of particles comprising hERG1a and SCN5A mRNAs and hERG1a protein per cell compared to the expected number based on chance using a maximum distance of 2 pixels between center of mass (minimum 50% overlap; mean ±SE; n = 13 cells, N = 2). (C) Histogram showing that 16% of hERG1a mRNA associate with hERG1a protein (actively translated population); of that percentage, 46% also interact with SCN5A transcripts (mean ±SE; n = 13 cells; N = 2). (D) Histogram showing the distribution of colocalized mRNA spots through the cytoplasm and from the nucleus revealing that RNP complexes are mostly localized within 10 µm from the nucleus. In the top right corner, representative examples of colocalized spots (yellow circles) and analysis of distance from the nucleus (white dashed arrows). 10.7554/eLife.52654.026Figure 5—source data 1.Association of hERG1a and SCN5A mRNAs with hERG1a protein raw data for Figure 5B.…

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  Figure 5—figure supplement 1.. hERG1a mRNA protein interaction. (A) Representative confocal images and enlargement (outlined in yellow) of iPSC-CMs subjected to Immunofluorescence combined with smFISH protocol showing the colocalization (yellow arrows) of hERG1a mRNA (magenta) and hERG1a protein (green). (B) Pie chart showing the percentage of hERG1a mRNA population interacting with hERG1a protein revealing that 16% of hERG1a mRNA were actively translated at the moment of fixation. 10.7554/eLife.52654.025Figure 5—figure supplement 1—source data 1.Association of hERG1a and SCN5A mRNAs with hERG1a protein raw data for Figure 5—figure supplement 1.

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  Figure 6.. Distribution and association of hERG1a and SCN5A transcripts under puromycin treatment in iPSC-CMs. (A) Representative confocal images and enlargement (outlined in yellow) of iPSC-CMs subjected to immunofluorescence combined with smFISH for control cells (left panel) or cells treated with 100 µM puromycin for 15 min (right panel). (B) The number of mRNAs detected per cell was plotted for SCN5A and hERG1a in the presence of puromycin and compared to control cells (lines represent mean ±SE). (C) Histogram showing the reduction of association between hERG1a mRNA and hERG1a protein after puromycin treatment compared to non-treated cells (mean ±SE). (D) Histogram showing that the % of triply colocalized particles (hERG1a protein or the ribosomal subunit S6 associated with both hERG1a and SCN5A mRNAs) is decreased upon puromycin treatment (mean ±SE). 10.7554/eLife.52654.030Figure 6—source data 1.Number of mRNA per cell after puromycin raw data for Figure 6B. 10.7554/eLife.52654.031Figure 6—source data 2.hERG1a and SCN5A mRNAs association raw data for Figure 6C. 10.7554/eLife.52654.032Figure 6—source data 3.hERG1a and SCN5A transcripts cotranslational association raw data for Figure 6D.